Ixeris dentata NAKAI Reduces Clinical Score and HIF-1 Expression in Experimental Colitis in Mice

Abstract

Ixeris dentata (ID) is an herbal medicine used in Asian countries to treat indigestion, pneumonia, hepatitis, contusions, and tumors; however, its effect on intestinal inflammation is unknown. Thus, we investigated the effect of ID in the dextran sulfate sodium (DSS) model of colitis in female BALB/c mice; animals were evaluated after seven days of DSS treatment. DSS-treated mice showed considerable clinical signs, including weight loss, reduced colon length, colonic epithelial injury, infiltration of inflammatory cells in the colon tissue, and upregulation of inflammatory mediators. However, administration of ID attenuated body weight loss, colon shortening, and the increase in disease activity index score. ID also significantly decreased the colonic mucosal injury and the number of infiltrating mast cells. Moreover, ID inhibited the expressions of cyclooxygenase-2 and hypoxia-inducible factor-1 α in colon tissue. Taken together, the results provide experimental evidence that ID might be a useful therapy for patients with ulcerative colitis.

Figure 1

The effect of Ixeris dentata NAKAI (ID) on dextran-sodium-sulfate- (DSS-) induced clinical signs. Ulcerative colitis was induced in female BALC/c mice by administering 5% DSS in the drinking water for seven days. Over the same period, ID (100 mg/kg) and the reference compound sulfasalazine (SFZ; 100 mg/kg) were given orally once daily. (a) Body weights were measured at the same time of the experimental days. (b) Colons were harvested on day 7, and colon lengths were measured. (c) Colon lengths in the four study groups. (d) Disease activity index scores in the four study groups. Values represent mean ± S.E.M. (n = 5). Data were analyzed by Student's t test (^# P < 0.05 versus control and *P < 0.05 versus DSS alone).

Figure 2

The effect of Ixeris dentata NAKAI (ID) on serum levels of interleukin- (IL-) 6 and tumor-necrosis-factor- (TNF-) α in DSS-induced colitis. Ulcerative colitis was induced by administering 5% DSS in the drinking water for seven days. Over the same period, ID (100 mg/kg) and the reference compound sulfasalazine (SFZ; 100 mg/kg) were given orally once daily. Cytokine production was determined by ELISA. (a) IL-6 production in mouse serum at day 7. (b) TNF-α production in mouse serum at day 7. (c) IL-6 production in colon tissue. (d) TNF-α production in colon tissue. Values represent mean ± S.E.M. (n = 5). Data were analyzed by Student's t test (^# P < 0.05 versus control and *P < 0.05 versus DSS alone).

Figure 3

The effect of Ixeris dentata NAKAI (ID) on dextran-sodium-sulfate- (DSS-) induced cyclooxygenase- (COX-) 2 and hypoxia-inducible-factor- (HIF-) 1α levels in colonic tissues. Ulcerative colitis was induced in female BALC/c mice by administering 5% DSS in the drinking water for seven days. Over the same period, ID (100 mg/kg) and the reference compound sulfasalazine (SFZ; 100 mg/kg) were given orally once daily. COX-2 and HIF-1α levels were determined by western blot analysis. (a) Representative western blot (of three independent experiments) of COX-2 and HIF-1α expression in colonic tissue. (b) Ratios of COX-2/GAPDH and (c) HIF-1α/GAPDH were determined by densitometry. (d) Sections of colons of DSS-treated mice with or without ID treatment were subjected to immunohistochemical analysis. Values represent mean ± S.E.M. Data were analyzed by Student's t test (^# P < 0.05 versus control and *P < 0.05 versus DSS alone).

Figure 4

The effects of Ixeris dentata NAKAI on epithelial injury and mast cells infiltration in dextran-sodium-sulfate- (DSS-) induced colitis. Ulcerative colitis was induced in female BALC/c mice by administering 5% DSS in the drinking water for seven days. Over the same period, ID (100 mg/kg) and the reference compound sulfasalazine (SFZ; 100 mg/kg) were given orally once daily. (a) Paraffin sections of colonic tissue were stained with hematoxylin and eosin (100x) or with toluidine blue for mast cell identification. Mast cell infiltration is indicated by the arrows (structures: TA, tunica adventitia; MM, muscularis mucosa; SL, submucosa layer; ML, mucosa layer; LU, lumen). Microscopic scores (b), thickness of mucosal layer (c), and number of mast cells (d) were presented. Values represent mean ± S.E.M. (n = 5). Data were analyzed by Student's t test (^# P < 0.05 versus control and *P < 0.05 versus DSS alone).

Figure 5

HPLC fingerprints of Ixeris dentata NAKAI (ID) and the standard 3,4-dihydroxy cinnamic acid. The mobile phase consisted of acetonitrile : distilled water : glacial acetic acid (15 : 85 : 1.5, isocratic manner). The injection volume was 10 μL of each sample, and flow rate was 1 mL/min. Wavelength was 254 nm. Retention time of 3,4-dihydroxy cinnamic acid was 14.3 min.